CN102687707A - Novel dasyhelea species and CO I (cytochrome c oxidase I) gene sequence thereof - Google Patents

Abstract

The invention discloses a novel dasyhelea species and a CO I (cytochrome c oxidase I) gene sequence thereof. The novel dasyhelea species belongs to dasyhelea and is named as dasyhelea arrhena Wang, Guan and Yu, sp. nov. The novel dasyhelea species is researched by methods of morphological identification, sequence analysis and the like, and results show that the novel dasyhelea species is novel in dasyhelea. In addition, a CO I gene sequence of the novel dasyhelea species is obtained, and basis for molecular identification of the novel dasyhelea species is provided.

Description

A kind of moth fly novel species and CO I gene order thereof

Technical field

The present invention relates to a moth fly, be specifically related to a kind of moth fly novel species and CO I gene order thereof.

Background technology

Moth fly is a miniature insect common in urban afforestation band and the gardens, belongs to diptera (Diptera) Heleidae (Ceratopogonidae) moth fly subfamily.So far as is known, the moth fly subfamily only comprise Psychoda ( DasyheleaKieffer, 1911) 1 genus.Because of moth fly right and wrong blood sucking midge class,, often get the brush-off to the person poultry harmless.But the effect of moth fly in propagating plant pollen and environmentally friendlyization reaction paid close attention to by people gradually.

For a long time; Evaluation to midge classes such as moth flys mainly relies on morphological feature to realize; Along with development of technology such as dna sequencings, people adopted molecular biology method that the characteristic DNA of midge class is studied successively but in recent years; In the hope of setting up new sorting technique, testify each other with the traditional classification method.

Mitochondria CO I gene is to use molecular labeling the most widely in the midge class identification research; Be mainly used in the Molecular Identification research of blood sucking midge at present; Successfully identify the species in a plurality of kinds of groups such as greyish black storehouse midge and apparent storehouse midge, can be applied in the Molecular Identification of moth fly equally.

Summary of the invention

The object of the present invention is to provide a kind of moth fly novel species and CO I gene order thereof.The diversity of world's species has been enriched in the discovery of this novel species, the acquisition of its CO I gene order, and the Rapid identification that can be these species provides scientific basis.

For realizing above-mentioned purpose, the present invention realizes through following technical scheme:

The gained sample is processed slide, under biomicroscope, observe, draw morphological feature figure, and consult relevant document both at home and abroad,, carry out species and identify according to the morphological feature of sample.This midge kind belongs to Psychoda, called after moth fly magnificent ( Dasyhelea arrhenaWang, Guan and Yu, sp. nov.).Type specimen is stored in Military Medical Science Institute medical insect sample shop.

Said moth fly novel species has following identification of morphology characteristic:

A, long 0.75 mm of ♂ wing, wide 0.24 mm;

B, body are brown, small-sized;

C, two compound eyes join, continuously distance.Pubescence is carefully small and dense between facet;

D, volume sheet are fusiformis;

E, feeler flagellum rag are obvious, and the wheel hair is sparse, the no distal process of the 15th joint, each joint long 19:10:10:10:10:10:10:10:12:22:23:19:21 of being that compares; AR 1.08;

F, antenna respectively save the long 5:6:15:10:6 that is that compares;

G, the nearly upper limb of chaperon have 2 in hair on the neck, each 3 of every sides;

H, the prosperity of cercoids the 9th backboard trailing edge pleurapophysis are embraced the acra joint and are the microbrachia shape, adeagus middle period complex structure, and wherein prominent sturdy, the end of a pair of pleurapophysis carefully curves inwardly; Carefully short in the paramere.

Adopt small insects minim DNA method for preparing template, through improved PCR reaction condition, by the CO I gene of synthetic this moth fly novel species of primer amplification, PCR product sequence is sent by professional biotech firm and is measured.Sequencing result passes through manual check and correction, sequence assembly, and in NCBI, carries out the Blast similarity searching, guarantees that institute's calling sequence is a target sequence.The CO I gene order SEQ ID No.1 (not containing primer) as follows of said moth fly novel species:

tttaatatta?ggagcccccg?atatagcttt?tcctcggata?aataatataa?gattttgaat 60

actccccccc?tctttatctt?tattactaat?tagtagctta?gtcgaaaatg?gtgccggaac 120

aggatgaact?gtttatccac?ctttagctag?taacatcgct?catagcgggg?catctgttga 180

tttggctatc?ttttctttac?atttagctgg?tatttcatca?attttagggg?cagtaaattt 240

tattactacc?attattaata?tacgatcaaa?tggaatctct?tttgatcgta?tacctctatt 300

tgtgtgatca?gtatttatta?cagctatcct?tttactgtta?tctctaccag?ttctagcagg 360

agcaattaca?atattattaa?cagaccggaa?tttaaataca?tcattttttg?accccgctgg 420

aggaggtgac?cctattttat?accaacatct?attttgattt?tttggacatc?ct 472?。

The PCR primer of the CO I gene of said moth fly novel species is characterized in that the PCR primer sequence is:

Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ';

Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '.

The method for identifying molecules of moth fly novel species of the present invention comprises:

1) separation and Extraction DNA from moth fly novel species tissue to be measured;

2) be that template is used a pair of primer with this DNA; Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 '; Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 ' goes out this moth fly novel species CO I gene through PCR amplification;

3) get an amount of step 2 then) the CO I gene that goes out of PCR amplification separate with agarose electrophoresis; Behind ethidium bromide staining, detect in gel imaging system; According to the big or small result of determination of amplified production,, then send biotech firm's order-checking if the ability specific amplification goes out the band of 524bp;

4) according to sequencing result, if with the autoploidy of said gene order SEQ ID NO.1 more than 98%, can judge that described tissue to be measured is moth fly magnificent.

The present invention can use the agarose gel electrophoresis detection method to detect amplification.

The present invention adopts traditional morphological to identify and combines with the molecular classification method, and said moth fly novel species is identified, can guarantee accuracy and reliability that species are identified.

Description of drawings

Fig. 1 is moth fly feeler figure magnificent of the present invention.

Fig. 2 is moth fly antenna figure magnificent of the present invention.

Fig. 3 is moth fly volume sheet figure magnificent of the present invention.

Fig. 4 is moth fly chaperon figure magnificent of the present invention.

Fig. 5 is moth fly cercoids magnificent of the present invention (going the adeagus middle period) figure.

Fig. 6 is moth fly adeagus middle period figure magnificent of the present invention.

Fig. 7 is that moth fly CO I gene PCR amplified production electrophoresis magnificent of the present invention is identified figure.Numbering is explained as follows: swimming lane 1-2 is the CO I gene of moth fly male worm magnificent, detects size and is the band of 524bp.M is the DNA marker of DL-2000.

Fig. 8 is that unknown moth fly CO I gene PCR amplified production electrophoresis is identified figure.Numbering is explained as follows: swimming lane 1 is the CO I gene of unknown moth fly male worm, detects size and is the band of 524bp.M is the DNA marker of DL-2000.

Embodiment

Embodiment 1

1, the collection of sample and preservation

Employing is waved the net method and is gathered, and adopts the sample that obtains and after freezing execution, directly is stored in 75% alcohol.

2, slide sample is made

Taking-up is stored in the sample in 75% alcohol, with dissecting needle head, sexual organ part and wing is downcut, and is convenient to film-making.Preparation of specimen carries out with reference to the described method in " Chinese Heleidae insect " (Yu Yixin chief editor, 2005).

The sample remainder moves in the PCR pipe, and 95% alcohol is preserved, one of every pipe, and carry out exclusive number, be used for DNA extraction.If need the long period to preserve, then should place-20 ℃ of refrigerators frozen the PCR pipe that the residue tissue site is housed.

3, dna profiling preparation

Adopt small insects minim DNA method for preparing template extract moth fly DNA magnificent (the Wen Lizhang chief editor. entomology research method and technological introduction [M]. Beijing: Science Press, 2010. 278-286), the DNA sample of extraction is subsequent use in-20 ℃ of preservations.

4, primer is synthetic

The present embodiment the primer is following:

Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 '

Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '

5, pcr amplification

The PCR reaction system of present embodiment is as shown in table 1.

Table 1 is a moth fly CO I gene PCR reaction system magnificent (50uL system)

Composition	Volume (uL)
		Template	10
Primer 1	1
		Primer 2	1
2×Mastermix	25
		ddH 2O	13

The response procedures of present embodiment is as shown in table 2.

Table 2 is a moth fly CO I gene PCR response procedures magnificent

Step	Reaction temperature (℃)	Time	Period
				Sex change in advance	94	3 min	-
Sex change	94	30 s	5
				Annealing	45	90 s	5
Extend	68	60 s	5
				Sex change	94	30 s	35
Annealing	51	90 s	35
				Extend	68	60 s	35
Extend	68	10 min	-

The PCR product is subsequent use in 4 ℃ of preservations.

6, pcr amplification product detects

Get 5 μ L pcr amplification products, with 1% agarose gel electrophoresis (120V voltage, electrophoresis 30 min).Ethidium bromide staining.Gel imaging system detects, and electrophoresis pattern is referring to accompanying drawing 7.

7, gene sequencing

Choose 2-3 effect preferably pcr amplification product send by order-checking (order-checking the primer identical) behind biotech firm's purifying with the PCR the primer, be the CO I gene order-SEQ ID No.1 of this moth fly novel species after the calibrated splicing of gained gene order.

Embodiment 2

1, the collection of sample and preservation

Employing is waved the net method and is gathered, and adopts the sample that obtains and after freezing execution, directly is stored in 75% alcohol.

2, sample chooses and and pre-treatment

From the midge class sample that collects, random choose goes out a moth fly, with dissecting needle head, sexual organ part and wing is downcut, and is convenient to film-making and carries out identification of morphology.Remainder moves in the PCR pipe, and 95% alcohol is preserved, one of every pipe, and carry out exclusive number, be used for DNA extraction.

3, dna profiling preparation

Adopt small insects minim DNA method for preparing template extract unknown moth fly DNA (the Wen Lizhang chief editor. entomology research method and technological introduction [M]. Beijing: Science Press, 2010. 278-286), the DNA sample of extraction is subsequent use in-20 ℃ of preservations.

4, primer is synthetic

The present embodiment the primer is following:

Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 '

Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '

5, pcr amplification

The PCR reaction system of present embodiment is as shown in table 3.

Table 3 is a unknown moth fly CO I gene PCR reaction system (50uL system)

Composition	Volume (uL)
		Template	10
Primer 1	1
		Primer 2	1
2 * Mastermix	25
		DdH 2O	13

The response procedures of present embodiment is as shown in table 4.

Table 4 is unknown moth fly CO I gene PCR response procedures

Step	Reaction temperature (℃)	Time	Period
				Sex change in advance	94	3 min	-
Sex change	94	30 s	5
				Annealing	45	90 s	5
Extend	68	60 s	5
				Sex change	94	30 s	35
Annealing	51	90 s	35
				Extend	68	60 s	35
Extend	68	10 min	-

The PCR product is subsequent use in 4 ℃ of preservations.

6, pcr amplification product detects

Get 5 μ L pcr amplification products, with 1% agarose gel electrophoresis (120V voltage, electrophoresis 30min).Ethidium bromide staining.Gel imaging system detects, and electrophoresis pattern is referring to accompanying drawing 8.The unknown moth fly of finding out embodiment 2 by electrophoretogram 8 also can specific amplification goes out the product of 524bp, can tentatively judge saidly should possibly be moth fly magnificent by the unknown moth fly.In order more clearly to prove is moth fly magnificent, willThe product of 524bp Send biotech firm's order-checking, test result shows and the autoploidy of gene order SEQ ID NO. 1 is 99.8%, thereby can confirm and should be moth fly magnificent by the unknown moth fly

Claims (5)

1. a moth fly novel species is characterized in that this midge kind belongs to Psychoda, called after moth fly magnificent ( Dasyhelea arrhenaWang, Guan and Yu, sp. nov.).

2. the described moth fly novel species of claim 1 is characterized in that having following identification of morphology characteristic:

A, long 0.75 mm of ♂ wing, wide 0.24 mm;

B, body are brown, small-sized;

C, two compound eyes join, distance continuously, and pubescence is carefully small and dense between facet;

D, volume sheet are fusiformis;

E, feeler flagellum rag are obvious, and the wheel hair is sparse, the no distal process of the 15th joint, each joint long 19:10:10:10:10:10:10:10:12:22:23:19:21 of being that compares; AR 1.08;

F, antenna respectively save the long 5:6:15:10:6 that is that compares;

G, the nearly upper limb of chaperon have 2 in hair on the neck, each 3 of every sides;

H, the prosperity of cercoids the 9th backboard trailing edge pleurapophysis are embraced the acra joint and are the microbrachia shape, adeagus middle period complex structure, and wherein prominent sturdy, the end of a pair of pleurapophysis carefully curves inwardly; Carefully short in the paramere.

3. the CO I gene of the described moth fly novel species of claim 1 has the gene order SEQ ID No.1 that is described below:

tttaatatta?ggagcccccg?atatagcttt?tcctcggata?aataatataa?gattttgaat 60

actccccccc?tctttatctt?tattactaat?tagtagctta?gtcgaaaatg?gtgccggaac 120

aggatgaact?gtttatccac?ctttagctag?taacatcgct?catagcgggg?catctgttga 180

tttggctatc?ttttctttac?atttagctgg?tatttcatca?attttagggg?cagtaaattt 240

tattactacc?attattaata?tacgatcaaa?tggaatctct?tttgatcgta?tacctctatt 300

tgtgtgatca?gtatttatta?cagctatcct?tttactgtta?tctctaccag?ttctagcagg 360

agcaattaca?atattattaa?cagaccggaa?tttaaataca?tcattttttg?accccgctgg 420

aggaggtgac?cctattttat?accaacatct?attttgattt?tttggacatc?ct 472?。

4. the PCR primer of the CO I gene of the said moth fly novel species of claim 3 is characterized in that the PCR primer sequence is:

Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ';

Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '.

5. the method for identifying molecules of a moth fly novel species comprises:

1) separation and Extraction DNA from moth fly novel species tissue to be measured;

2) be that template is used a pair of primer with this DNA; Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 '; Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 ' goes out this moth fly novel species CO I gene through PCR amplification;

3) get an amount of step 2 then) the CO I gene that goes out of PCR amplification separate with agarose electrophoresis; Behind ethidium bromide staining, detect in gel imaging system; According to the big or small result of determination of amplified production,, then send biotech firm's order-checking if the ability specific amplification goes out the band of 524bp;

4) according to sequencing result, if with the autoploidy of said gene order SEQ ID NO.1 more than 98%, can judge that described tissue to be measured is moth fly magnificent.

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